PROJECT SUMMARY Lung cancer is the leading cause of cancer-related death in the United States. About 90% of lung cancer cases are associated with genetic/epigenetic changes induced by tobacco smoke (TS). Although all types of lung cells could be affected by TS, the effect on stem cells is particularly alarming owing to their longevity and propensity for transformation. Therefore, selective targeting of altered stem cells known as cancer stem cells (CSCs) could prevent the development of lung cancer. In this application, we will test the hypothesis that targeting of uniquely tumorigenic putative CSCs expressing high levels of aldehyde dehydrogenase 1(ALDH1H) and CD44 (CD44H) with the combination of the repurposed drugs disulfiram (DSF) and sulfasalazine (SAS) which are nano-formulated to enhance bioavailability will suppress the development and progression of carcinogen-induced and spontaneous lung tumor in mice. These hypotheses will be tested by the following three specific aims: Specific Aim 1: Determine the efficacy of SLN-DSF, SLN-SAS and SLN-DSF+SAS to suppress NNK- or mutant K-ras induced lung tumorigenesis by targeting ALDH1HCD44H subpopulations of lung cells. In this aim, mice treated with NNK or harboring mutations in K-ras gene will be given the drugs and modulation of tumor burden, frequency of ALDH1HCD44H lung tumor cells and CSC-associated proteins will be analyzed. Specific Aim 2: Determine the immunosuppressive effects of ALDH1HCD44H putative LCSCs and whether the anti-cancer effects of SLN-DSF-SAS are mediated, at least in part, via immunomodulatory mechanism and its potential to enhance the therapeutic efficacy of anti-PD-L1 immune checkpoint inhibitors. Hypothesis: Overexpression of PD-L1 by ALDH1HCD44H lung tumor cells endows them immunosuppressive properties and modulation of these properties by SLN-DSF-SAS could potentiate anti-PD-L1-induced rescuing of dysfunctional cytotoxic T cells and tumor destruction. Specific Aim 3: Determine the role of common NSCLC genetic alterations in the generation, proliferation, self- renewal, and tumor propagating efficiency of ALDH1HCD44H putative CSCs and if these effects are modulated by SLN-DSF+SAS. Hypothesis: The genotype of transformed lung cells could be an important determinant of the self-renewal and tumor-propagating potential of ALDH1H CD44H fractions. Impact: Targeting ALDH1H CD44H putative CSCs is a new paradigm shift in lung cancer prevention and treatment as these cells are believed to be the cell of origin of cancer.